Trauma-hemorrhage (T-H), a physical stress, causes inflammatory response in several organs and tissues (1, 2), but its effect on the central nervous system (CNS) has been relatively unexplored. The CNS senses inflammation in the body through circulating cytokines after infection or injury to peripheral tissues (3). Proinflammatory cytokines may promote the development of stress response, including the hypothalamic-pituitary-adrenal (HPA) axis (4), and excessive inflammation can result in a fatal outcome (5). However, the precise mechanisms by which the cytokine signal is conveyed centrally are still unclear. In this regard, injury of peripheral tissues induces an increase in brain cytokine synthesis, which seems to play a particularly prominent role in coordinating centrally mediated responses to stress, such as tail shock in rats (6) and acute myocardial ischemia (7).
Microglial cells, the resident brain macrophages, are normally present in the adult brain in a resting state (8), and their activation is characterized by secretion of a wide spectrum of proinflammatory and cytotoxic molecules, which can sustain inflammation and mediate neuronal damage. Recent study has shown that microglial cells are involved in stress-induced cytokine production (9). We therefore hypothesized that T-H activates microglial cells, which cause proinflammatory cytokine synthesis and release within the brain, leading to the development of stress response under those conditions.
The gonadal steroid 17β-estradiol (E2), a female sex hormone, produces anti-inflammatory effects in peripheral immune cells, such as Kupffer cells after T-H (10). In addition, E2 serves as a central anti-inflammatory molecule that acts on glial cells, including microglial cells (11). On the basis of this information, we examined the effects of E2 within the brain on microglial cell-mediated inflammatory response to T-H. We measured tumor necrosis factor (TNF-α) as an indicator of the general state of proinflammatory cytokine synthesis because it appears early in the proinflammatory cytokine cascade (12). Furthermore, minocycline, which inhibits microglial activation (13), was used to determine whether inflammatory response in the brain is caused by microglial cells.
MATERIALS AND METHODS
All procedures were performed in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals; the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham approved the experimental protocol.
Experiments were performed on adult male Sprague-Dawley rats (weight, 275-325 g; Charles River Labs, Wilmington, Mass) that were maintained on a 12:12-h light:dark cycle (lights on at 0600 h) with food and water available ad libitum.
Our previously described nonheparinized model of T-H and resuscitation in the rat was used with minor modifications (14). Briefly, rats were fasted overnight but allowed access to water ad libitum before surgery. After anesthesia by isoflurane (Attane; Minrad, Bethlehem, Pa) inhalation, soft tissue trauma (5-cm ventral midline laparotomy) was performed, and the abdominal wound was closed in layers. Catheters (polyethylene, PE-50 tubing; Becton-Dickinson, Franklin Lakes, NJ) were placed in both femoral arteries and right femoral vein. The left arterial catheter was connected to a transducer and a blood pressure analyzer (Digi-Med, Louisville, Ky) to monitor blood pressure throughout the experiment. The right femoral arterial catheter was used for bleeding. The right femoral vein catheter was used for drug or Ringer's lactate (RL) injections. All incisions were bathed with 1% lidocaine (Elkins-Sinn, Cherry Hill, NJ) to provide analgesia throughout the experiments. Rats were allowed to awaken and were rapidly bled to a MAP of 35 to 40 mmHg (i.e., severe hypotension) within 10 min. The rapid bleeding on awakening puts the animals in a state of depressed sensibility, thus minimizing distress to the animals. The blood pressure was maintained until rats could not maintain MAP of 35 mmHg unless extra fluid, in the form of RL, was administered. After the maximum bleedout, MAP was maintained between 35 and 40 mmHg until 40% of the maximum bleedout volume was returned in the form of RL. Rats were then resuscitated with four times the volume of the withdrawn blood over 60 min with RL. The sham-operated rats underwent the same surgical procedure but were neither bled nor resuscitated.
Treatment 1: 17β-estradiol
T-H and sham rats were treated with E2 (1 mg/kg body weight [BW]; Sigma, St Louis, Mo) or vehicle (VEH, cyclodextrin, 20 mg/kg BW; Sigma) intravenously at the beginning of resuscitation. Sham rats received injection of either VEH or E2 at the time of sham operation.
Treatment 2: microglia inhibition
Minocycline is a highly lipophilic tetracycline that crosses the blood-brain barrier easily (15). Minocycline has been widely used to block central inflammatory response and seems to have effects that are quite specific to actions on microglia (13). T-H and sham rats were pretreated with minocycline (40 mg/kg BW; Sigma) or equivalent volume of VEH intraperitoneally (i.p.) 1 h before surgery. This method was chosen because Blandino et al. (9) recently showed that 40 mg/kg i.p. completely abolished the expression of cytokines in the hypothalamus after foot shock.
Blood and tissue sampling
At the end of resuscitation, the animals were anesthetized with isoflurane, and blood was obtained via cardiac puncture using a syringe coated with heparin (Abraxis Pharmaceutical Production, Schaumburg, Ill). This was immediately followed by perfusion of saline containing heparin, and brains were collected. Blood was centrifuged (2,500g, 10 min, 4°C), and plasma was removed and stored at −80°C until assayed for TNF-α.
Collection of brain tissue
After the brain tissue was removed from the skull, the hypothalamus was removed as described previously (16). This was carried out using the posterior part of the optic chiasm as the anterior limit, the anterior part of the mammillary bodies as the posterior limit, and the lateral hypothalamic sulci as the lateral limit. The frontal cortex and brain stem were also taken and flash frozen. All tissue was stored at −80°C until the time of assay for TNF-α.
Microglia isolation and flow cytometry
Microglia from rats was harvested using a Percoll gradient as previously described (17). Rats were transcardially perfused at the end of resuscitation, and brains were collected in 10 mL HBSS per brain, containing 0.05% collagenase D (Roche, Indianapolis, Ind), 0.1 mg/mL N-tosyl-l-leucine chloromethyl ketone (Sigma), 10 mg/mL dispase (Roche), and 10 mM HEPES buffer (Invitrogen, Carlsbad, Calif). Brain tissues were dispersed with a glass Dounce homogenizer, and cells were separated over discontinuous Percoll gradients. For culture experiments, cells were grown on collagen-coated dishes in phenol red-free RPMI 1640 medium supplemented with charcoal-stripped (steroid deficient) sera. For cytokine measurement in the supernatants, cells were seeded at a density of 2 × 105 cells per well after isolation and incubated for 24 h. For flow cytometry after isolation, the cell suspensions were first blocked with 0.5 μg/mL purified anti-rat CD32 Fc blocking antibody (Ab; BD Pharmingen, San Diego, Calif) for 15 min on ice. Cells were then washed with staining buffer two times, then stained with Ab against surface markers, Phycoerythrin (PE)-conjugated anti-rat CD11b/c (BD Pharmingen), and fluorescein isothiocyanate-conjugated anti-rat CD54 (BD Pharmingen) for 45 min. After two washes in the staining buffer, cells were resuspended in 200 μL of staining buffer and analyzed using the LSRII flow cytometer (BD Biosciences, San Jose, Calif). Isotype-matched immunoglobulin Gs were used as a nonspecific staining control. FITC or PE conjugated anti-rat Abs were incubated with CompBeads (BD Biosciences), respectively, which subsequently provide distinct positive and negative stained populations; these were used as positive/negative controls to set compensation levels.
Levels of TNF-α in plasma, tissue, and cell-free media (cells were seeded at a density of 2 × 105 cells/well for 24 h, and supernatant was then collected) were determined using a rat-specific enzyme-linked immunosorbent assay (ELISA, R&D Systems, Minneapolis, Minn) according to the manufacturer's instructions.
Data were presented as mean ± SE (n = 4-5 rats/group). ANOVA followed by a Tukey-Kramer test was used to compare the treatment effects of E2 between groups. P < 0.05 was considered statistically significant.
Anti-inflammatory effects of E2 on plasma and brain tissues after T-H
T-H significantly increased plasma TNF-α levels at the end of resuscitation, and E2 prevented T-H-induced plasma cytokine levels (Fig. 1A), as we have previously reported (18). Since recent study has shown that acute myocardial infarction induces hypothalamic proinflammatory cytokines (7), we hypothesized that T-H also induces proinflammatory cytokines in the brain, which affects neural systems in the CNS. The results show that increased TNF-α in brain after T-H was observed in the hypothalamus (Fig. 1B) and brain stem (Fig. 1C) but not in the cortex (Fig. 1D). E2 administration after T-H did not produce any effect on brain tissue TNF-α levels in sham rats. In T-H rats treated with E2, the increased TNF-α levels after T-H in the hypothalamus were significantly decreased (P < 0.05) compared with VEH administration. The increased TNF-α levels induced by T-H in the brain stem were also decreased by E2 treatment.
E2 inhibits T-H-induced activation of hypothalamic microglial inflammatory functions
We also examined microglial cells, the resident brain macrophages, to determine whether microglial cells are involved in the brain cytokine synthesis after T-H. In the CNS, microglial cells are believed to be the primary cellular sources of TNF-α expression (19), and activated microglial cells enhance intracellular adhesion molecule 1 (ICAM-1) expression (20). We therefore hypothesized that T-H activates microglial cells to produce TNF-α within the brain. As shown in Figure 2A, we gated [CD11b/c]high positive microglial cells and examined that population for CD54 or ICAM-1 expression as activated microglial cells by flow cytometry. The increase in CD54 staining on [CD11b/c]high microglial cells was apparent after T-H at the end of resuscitation compared with sham groups. However, the increase in CD54 staining was significantly inhibited by E2 treatment in vivo (Fig. 2B).
Central proinflammatory cytokine production is independent of circulating proinflammatory cytokines after T-H
To examine whether central proinflammatory cytokines are produced by microglial cells and whether anti-inflammatory effects of E2 on the central inflammatory response are mediated by directly inhibiting microglial cells after T-H, we injected animals i.p. with minocycline, a microglia inhibitor, 1 h before T-H. We measured plasma and hypothalamic tissue levels of TNF-α to test the anti-inflammatory effects of minocycline at the end of resuscitation. In addition, we measured TNF-α production capacity of microglial cells isolated from brain after T-H and compared anti-inflammatory effects of minocycline with E2 treatment. Pretreatment with minocycline markedly blocked the T-H-induced increase in hypothalamic TNF-α levels (Fig. 3A). In contrast, minocycline had no effect on plasma TNF-α levels compared with T-H VEH rats (Fig. 3B), whereas E2 attenuated T-H-induced increase in plasma TNF-α levels (Fig. 1A). ELISA of TNF-α secretion in the supernatants of microglia after 24 h culture indicated a marked increase in TNF-α production capacity of microglial cells in T-H receiving VEH compared with sham. E2 significantly attenuated T-H-induced increase in TNF-α productive capacity of microglial cells; however, the inhibitory effect of minocycline on TNF-α productive capacity of microglial cells was higher than E2 after T-H (Fig. 3C).
We hypothesized that microglial cells are the primary cellular source of proinflammatory cytokines in the CNS after T-H, and E2 administration after T-H provides a central anti-inflammatory effect under those conditions by acting on microglial cells. Our results show that T-H induces a significant increase in hypothalamic TNF-α levels as well as TNF-α production by isolated microglial cells, and E2 administration after T-H significantly prevented T-H-induced hypothalamic TNF-α levels as well as TNF-α production by isolated microglial cells (Fig. 4). These findings provide new important evidence of T-H-induced central proinflammatory cytokine response mediated by microglial cells and also that E2 plays an anti-inflammatory role in this microglial cell-mediated activation of inflammatory function in the brain after T-H.
Neuroimmunology is a rapidly growing research field that seeks to define the interactions between the immune system and the nervous system. In this regard, Cooney (21) indicated that cytokine-mediated JAK/STAT signaling controls a number of important biological responses, including immune function. With respect to the CNS, brain injury can lead to the production of inflammatory mediators in the CNS, such as TNF-α (22). Furthermore, Francis et al. (7) suggested that neural signaling mechanism, including cardiac sensory afferent fibers, mediates this central inflammatory response to myocardial infarction. Our results show for the first time that there are some differences in TNF-α expression in response to T-H in different regions of the brain. This notion is in accordance with a previous report which showed that the distribution of neurotransmitter receptor is different in different regions of the brain (23). However, it remains to be determined if and how TNF-α expression changes with time in the brain after T-H. In contrast to nontraumatic brain injury as used in our study, Ahn et al. (24) have shown that brain IL-1β expression was different compared with TNF-α under those conditions. Further studies are therefore needed to determine how the responses of other proinflammatory cytokines such as IL-1β and IL-6 are in the hypothalamic region after T-H and resuscitation and whether E2 has similar salutary effects on those cytokines.
The hypothalamic region of the brain is a center for stress response. It contributes substantially to integrate the activation of the HPA axis and the neuroendocrine control of visceral autonomic functions (25). Because the immune and the nervous systems are linked functionally and anatomically and because they influence each other and interact with each other, we propose that hypothalamic functions such as the HPA axis may be involved in modulation of local cytokine release from immune cells within the brain after T-H. Future work will be required to determine the immune and the neuronal interaction in the central inflammatory responses to T-H, particularly in the hypothalamus.
Microglial cells are well known to be the primary cellular source of TNF-α expression in the CNS (19). On the basis of our observations that T-H induced increased TNF-α within the brain, we hypothesized that microglial cells are involved in this central inflammatory response early after T-H. Previous study has shown that microglial cells are rapidly activated and are responsible for a number of immunological and other stress events in the brain (26). Furthermore, ICAM-1 has been shown to be expressed on activated microglial cells in chronic and acute CNS disorders (27), and microglial activation is associated with increased ICAM-1 expression (20). Our study shows that T-H significantly increased ICAM-1 expression in microglial cells, and administration of E2 resulted in the down-regulation of T-H-induced increase in ICAM-1 expression, indicating that E2 has direct effects on microglial cells in the brain by inhibiting their activation early after T-H. In this regard, our previous study has also shown that ICAM-1 expression in liver, intestine, and lung tissues is increased after T-H (28); however, administration of E2 after T-H down-regulated ICAM-1 expression in these tissues under those conditions. Thus, the present results confirm previous findings and demonstrate that the same also occurs in the microglia after T-H with and without E2 treatment.
Although ICAM-1 expression in the normal brain is restricted to vascular endothelial cells, some forms of neural injury cause a rapid induction of this molecule on a number of cell types in the affected CNS, including microglial cells (29). In fact, activated microglial cells rapidly adhere to the surface of damaged neurons (30), and ICAM-1 is expressed on components of the CNS, including microglia (29). However, cell surface antigens such as complement receptors and MHC class II molecules are also up-regulated on activated microglial cells (26). In addition, acute neurological disorders, such as stroke, brain trauma, or inflammation, elicit rapid activation of microglial cells with morphologic changes from ramified into ameboid cells (31). In this study, activation of microglial cells after T-H was monitored by changes in CD54 expression by flow cytometry; however, different markers for detection of microglial activation should be examined to determine their function after T-H in future studies.
We found the induction of TNF-α in the CNS concomitant with plasma TNF-α level early after T-H; however, the relationship between brain and circulating cytokines remains unclear. There are some communication pathways between the peripheral immune response and the brain, such as neural and humoral routes (3). Accordingly, we also hypothesized that T-H-induced central TNF-α observed within the brain is mainly derived from activated microglial cells and, thus, is not blood borne. Minocycline, a second-generation semisynthetic tetracycline with broad spectrum antimicrobial activity (13), effectively crosses the blood-brain barrier (15). It also prevents microglial activation and is neuroprotective in a model of cerebral ischemia (13) and traumatic brain injury (32). We therefore compared the anti-inflammatory effects of minocycline with E2 on TNF-α synthesis and release both in plasma and hypothalamus. Our results showed that pretreatment with minocycline completely blocked the hypothalamic TNF-α response to T-H, whereas the circulating TNF-α response was unaffected in the same animals. This indicates that microglial cells are the main source of brain TNF-α levels and that blood contribution to the tissue content of TNF-α protein, at least as far as the hypothalamus, is unlikely early after T-H, consistent with a previous report (6). Taken together, the increase seen in hypothalamic TNF-α due to T-H seems to be centrally driven and not by blood-borne cytokine signaling.
TNF-α production of microglial cells isolated from brain was measured to examine the cellular response of microglia after T-H. In this regard, it is well known that activated microglial cells produce proinflammatory mediators such as chemokines and cytokines (26) including TNF-α (19). However, astrocytes, a type of glial cell, also become activated and produce a variety of factors including cytokines (33). In this study, pretreatment with minocycline in vivo significantly prevented T-H-induced increase in TNF-α production by isolated microglial cells. In addition, the TNF-α levels in the supernatants were almost the same level as sham groups, consistent with hypothalamic tissue levels (Fig. 3B). Although we cannot exclude the possibility that astrocytes also participate in central inflammatory response to T-H, these results indicate that microglial cells are the primary cellular source of TNF-α expression within the brain, at least in the early stage after T-H.
In addition to its role as a sex hormone, E2 has important anti-inflammatory as well as immunomodulatory effects on macrophage and lymphocyte functions after T-H (34). Our previous study indicates that Kupffer cells are the major source of cytokines including TNF-α and contribute to the increased circulating cytokine levels after T-H (2). In addition, the salutary effects of E2 on Kupffer cell functions are mediated predominantly via estrogen receptor α (ER)-α (10). With regard to microglia, expression of ER is observed in microglial cells (35), and our results showed that E2 significantly prevented T-H-induced increase in TNF-α production by isolated microglial cells. These results are consistent with the finding that a major anti-inflammatory activity of E2 in microglia is evident even when microglia cells are stimulated with LPS (36). Furthermore, Vegeto et al. (11) have shown that the anti-inflammatory effect of E2 in microglial cells is mediated by TNF-α. Thus, one possibility is that E2 may be involved in the modulation of microglial cell-mediated inflammatory response through TNF-α after T-H. Our previous study has shown that the effect of E2 in reducing hepatic injury after T-H is mediated through G protein-coupled receptor 30 (GPR 30) (37). GPR 30 has been detected in the hypothalamus as the nongenomic ER (38). Additional studies examining the cellular expression of ER or GPR 30 on microglia will be necessary to conclusively implicate microglia as the cellular target and the specific receptor for producing the salutary effects of E2 after T-H.
T-H results in a profound suppression of cell-mediated immune response (39, 40). This is accompanied by an augmented release of anti-inflammatory TH2 cytokines, which contributes to the depressed immune response after T-H. In addition, TNF-α productive capacity of macrophages harvested from liver and lung was significantly increased after T-H, whereas cytokine production by peripheral blood mononuclear cells and splenic and peritoneal macrophages was markedly decreased under the same experimental conditions (39, 41). The CNS microglia forms an innate immune system that has the potential to initiate immune responses to stress within that system. These processes are usually considered to be harmful, and microglia seem to be the most capable to initiate and sustain a T-cell-mediated immune response (42). Our results showing that E2 directly blocks activation of microglial inflammatory response suggest that E2 has a tissue compartment-specific role in mediating the protective effects on organs and immune cell functions even in the CNS.
In conclusion, our results demonstrate that T-H significantly induced tissue TNF-α levels in some regions of brain, which directly correlated with increased expression of activated microglial cells as well as TNF-α production by isolated microglial cells at the end of resuscitation. By acting on microglia, E2 inhibits microglial cell-mediated central inflammatory response to T-H. For this reason, E2 could be an ideal anti-inflammatory hormone for curtailing central inflammatory response in the early phase of T-H.
The authors thank Dr Chi-Hsun Hsieh for his help with flow cytometry and analysis, Dr William J Hubbard and Dr Raghavan Raju for many helpful discussions, and Bobbi Smith for her assistance in preparing this manuscript.
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